Our long term goal is to understand the biology, the biochemistry and the genetics of how cells protect themselves against the deleterious effects of DNA damage, with particular emphasis upon the mechanisms cells employ to protect against DNA damage derived from endogenous cellular compounds. For the most part our studies involve investigating how cells protect themselves against DNA alkylation damage, but we will also examine how cells protect themselves against DNA oxidative damage. The mechanisms that cell employ to defend against DNA damaging agents have been highly conserved, and we now know that bacteria yeast and human cells employ similar strategies to repair DNA damage. Our research in the last grant period led us into studies on spontaneous mutation in both prokaryotes and eukaryotes; further studies on spontaneous mutation in eukaryotes is the main focus of this application. The plan was designed to encompass research at various levels of development; i.e., from further characterizing pathways already known to affect spontaneous mutation to identifying new pathways that affect spontaneous mutation. This purposefully broad-based plan results in a diverse proposal which I believe is compatible and complementary to-the other projects in this program, and which I believe will enhance our understanding of spontaneous mutation in eukaryotes. The specific Aims include the following: explore the role that DNA repair methyltransferases play in limiting spontaneous mutation; determine whether the natural metabolite S-adenosylmethionine produces spontaneous DNA damage that can lead to spontaneous mutations; explore the role that 3-methyladenine DNA glycosylases enzymes play in generating spontaneous abasic sites which can lead to spontaneous mutation; explore the proposed interaction between the DNA mismatch repair pathway and DNA alkylation damage; isolate and characterize new S. cerevisiae mutator strains that are compromised in their ability to deal with oxidative damage to DNA and DNA precursors.